Bactericidal and Virucidal Activity of Povidone-Iodine and Chlorhexidine Gluconate Cleansers in an In Vivo Hand Hygiene Clinical Simulation Study

Bactericidal and Virucidal Activity of Povidone-Iodine and Chlorhexidine Gluconate Cleansers in... Infect Dis Ther (2018) 7:235–247 https://doi.org/10.1007/s40121-018-0202-5 ORIGINAL RESEARCH Bactericidal and Virucidal Activity of Povidone-Iodine and Chlorhexidine Gluconate Cleansers in an In Vivo Hand Hygiene Clinical Simulation Study . . . Maren Eggers Torsten Koburger-Janssen Lois S. Ward Craig Newby Stefan Mu ¨ ller Received: February 6, 2018 / Published online: May 14, 2018 The Author(s) 2018 method for bacteria), which was adapted for ABSTRACT virucidal testing. Methods: Separate test series were performed Introduction: Standard in vitro and in vivo for bactericidal (Escherichia coli) and virucidal tests help demonstrate efficacy of hand hygiene [murine norovirus (MNV)] testing. After pre- products; however, there is no standard in vivo washing and artificial contamination of hands test method for viruses. We investigated the with test organisms, volunteers underwent bactericidal and virucidal efficacy of povidone- testing with 3 and 5 mL of each product for iodine (PVP-I) 7.5% scalp and skin cleanser, contact times of 15, 30 and 60 s according to a chlorhexidine gluconate (CHG) 4% hand Latin-square randomization. The number of test cleanser and the reference hand wash (soft soap) organisms released from fingertips into sam- in 15 healthy volunteers following European pling fluids was assessed before and after hand Standard EN1499 (hygienic hand wash test washing and mean log reduction factor (RF) was calculated. RFs (test-reference) were com- Enhanced Digital Features To view enhanced digital pared using a Wilcoxon–Wilcox multiple com- features for this article go to https://doi.org/10.6084/ parisons test per EN1499; efficacy was m9.figshare.6200894. concluded if p B 0.01. Results: PVP-I 7.5% and CHG 4% cleansers M. Eggers both passed EN1499 requirements against Labor Prof Gisela Enders MVZ GbR, Stuttgart, E. coli, with statistically significantly greater (p Germany B 0.01) mean log RFs compared with refer- T. Koburger-Janssen ence soft soap across all tests (PVP-I: 4.09–5.27; Hygiene Nord GmbH, Greifswald, Germany CHG: 4.12–5.22; soap: 2.75–3.11). The experi- L. S. Ward mental design using EN1499 was applicable to Clinical Operations, Mundipharma Research testing with MNV as discriminatory and repro- Limited, Cambridge, UK ducible results were generated. Mean log RFs C. Newby of MNV were statistically significantly greater R&D, Mundipharma Manufacturing Pte Ltd, for PVP-I (1.57–2.57) compared with soft soap Singapore, Singapore (1.24–1.62), while mean log RFs with CHG S. Mu ¨ ller (&) (0.90–1.34) were lower than for soft soap across Pharmacologicial and Translational Science, all tests. Mundipharma Research GmbH & Co KG, Limburg, Conclusion: PVP-I 7.5% cleanser showed supe- Germany rior efficacy against MNV compared to soft soap e-mail: stefan.mueller@mundipharma-rd.eu 236 Infect Dis Ther (2018) 7:235–247 and CHG 4% cleanser, while both PVP-I and effective than plain soap at removing viruses in CHG were superior to soft soap against E. coli. all tests. The adapted test method using murine The experimental set-up may be applicable to norovirus worked well, giving consistent results future testing for antiviral hand washes. for each product, and may be suitable for future Funding: Mundipharma Manufacturing Pte testing of antiseptic products against viruses. Ltd. Plain Language Summary: Plain language INTRODUCTION summary available for this article. The importance of hand hygiene in health care Keywords: Antimicrobial wash; Bactericidal and community settings is undoubted, with activity; Chlorhexidine gluconate; Escherichia numerous studies demonstrating the associa- coli; Hand-cleansing agents; Hand hygiene; tion between proper hand hygiene and reduc- Infection control; Non-enveloped virus; tions in both rates of nosocomial infections Povidone-iodine; Virucidal activity [1–3] and rates of infectious illnesses in com- munities [4]. Hand hygiene is also an infection control procedure that can be rapidly adopted PLAIN LANGUAGE SUMMARY by the public and healthcare workers in the case of emerging and re-emerging infectious diseases Serious infectious disease outbreaks and hospi- to limit the spread of viruses by breaking the tal-acquired infections are threats to human transmission [5, 6]. health. Effective vaccines and drug products are Multiple hand hygiene agents are currently often not available, but antiseptic products for available including plain non-medicated soap, hand washing can help to control the spread of medicated hand washes with various active disease. Standard methods are used to test the ingredients, and waterless or alcohol-based effectiveness of hand hygiene products in the hand rubs. Although the use of alcohol-based laboratory and on the hands of human hand rubs is convenient, the World Health volunteers. Organization (WHO) recommends washing We used a standard hand washing test hands with soap and water when visibly dirty or method to find out whether two antiseptic visibly soiled with blood or other body fluids products work against bacteria (Escherichia coli). [2]. It is recognized that some antiseptics and The method was adapted to also test effective- alcohol-based hand rubs are characterized by ness against viruses (murine norovirus). The resistant bacteria and efficacy gaps [1–3]. products tested were povidone-iodine scalp and In vitro suspension tests and human challenge skin cleanser, and chlorhexidine gluconate trials help to demonstrate which hand hygiene hand cleanser, which were compared to plain agents are fit for purpose, but variations in soft soap. After contamination of hands with methodology affect the measurement of effi- the bacteria/virus, volunteers washed their cacy [7]. Ensuring that hand rub and hand wash hands for 15, 30 or 60 s, using 3 or 5 mL of products pass standardized antimicrobial activ- product, in separate tests. Bacteria/viruses pre- ity tests helps to better understand and interpret sent on volunteers’ hands were measured before efficacy results [7]. and after hand washing. The antiseptic product In Europe, the most commonly used meth- had to remove significantly more bacteria/ ods to test hand antiseptics are those of the viruses than plain soap to be rated as effective. European Committee for Standardization Povidone-iodine cleanser and chlorhexidine (CEN), while in the USA and Canada, the stan- gluconate cleanser were both more effective dards of ASTM International (formerly, the than plain soap at removing bacteria from vol- American Society for Testing and Materials) are unteers’ hands. Povidone-iodine cleanser was used. The CEN has adopted a hierarchical, sys- more effective than plain soap at removing tematic approach of product testing. In this viruses, except when using 3 mL product for concept, European Standards EN1276 and 15 s. Chlorhexidine gluconate cleanser was less Infect Dis Ther (2018) 7:235–247 237 EN14476 are established initial in vitro methods methicillin-resistant Staphylococcus aureus to determine the bactericidal and virucidal (MRSA), Acinetobacter baumannii and E. coli from efficacy of antiseptics and disinfectants [8, 9]. volunteers’ hands, while CHG was only signifi- The next step in the CEN testing scheme is cantly more effective than plain soap at European standard EN1499 (for hand washes) removing E. coli [30–33]. In vivo virucidal and EN1500 (for hand rubs), each Phase 2/Step studies are generally lacking; however, in a 2 hand simulation studies where the hands of modified finger pad test based on the ASTM E volunteers are artificially contaminated with 1838 [34] using MNV, PVP-I was shown to have E. coli and the test product is compared against a superior efficacy to three alcohol-based hand reference procedure in a crossover design rubs while 4% CHG and 1% triclosan showed [10, 11]. To fill in gaps within the in vitro CEN no activity [35]. testing, modified vaccinia virus Ankara (MVA) We undertook two hand hygiene in vivo was introduced in 2015 as a reference virus for clinical simulation studies to investigate the the claim of virucidal activity against enveloped bactericidal and virucidal efficacy of PVP-I 7.5% viruses for hygienic hand rub and hand wash scalp and skin cleanser and CHG 4% hand products [9, 12, 13]. However, it has not been cleanser versus the reference hand wash (soft possible to develop a Phase 2/Step 2 test for soap) on healthy volunteers’ hands. Both stud- enveloped viruses due to the lack of availability ies followed European standard EN1499 as a of a safe enveloped virus that can be ethically function of employed soap volume and hand used in testing on volunteers’ hands, and there washing time. The virucidal study was per- is currently no standard in vivo test method for formed in an exploratory manner using murine viruses. To fill this gap, a hand simulation test norovirus (MNV) as model test virus. has recently been considered that uses murine norovirus (MNV), a non-pathogenic (biosafety METHODS level 1) non-enveloped virus, which is more resilient to antiseptics than enveloped viruses, Study Design as a model test organism. Povidone-iodine (PVP-I) and chlorhexidine Each study was randomized, controlled, open gluconate (CHG) are broad-spectrum antimi- crobials that have been used in infection con- label, crossover and exploratory in design. The bactericidal study was conducted at the labora- trol and prevention for more than 60 years. PVP-I has well-established general antimicrobial tories of Hygiene Nord, Greifswald, Germany, from 1 September 2016 to 19 January 2017. The activity, particularly in relation to resistant organisms [2], demonstrating in vitro activity virucidal study was conducted at the laborato- ries of Labor Prof. Gisela Enders MVZ GbR, against Gram-positive, Gram-negative and some spore-forming bacteria (Clostridia, Bacillus Stuttgart, Germany, from 14 September 2016 to spp.) and mycobacteria [2, 14–18] and a wide 12 December 2016. Hand hygiene tests were performed on the hands of healthy volunteers range of enveloped and non-enveloped viruses [12, 13, 19–22]. In contrast, CHG has good simulating practical use according to the methods described in European Standard in vitro activity against Gram-positive bacteria and enveloped viruses, less activity against EN1499:2013 (hygienic hand wash test method for bacteria), which were adapted and used in Gram-negative bacteria and non-enveloped viruses, and minimal activity against mycobac- an exploratory manner to assess the virucidal activity of the hand cleansers. All procedures teria [22–28]. CHG resistance is a concern and has been detected in isolates of Enterobacter spp., performed in studies involving human partici- pants were in accordance with the 1964 Hel- Pseudomonas spp., Proteus spp., Providencia spp. sinki declaration and its later amendments or and Enterococcus spp [29]. In vivo studies using artificial fingertip contamination have previ- comparable ethical standards. Informed con- sent was obtained from all individual partici- ously shown PVP-I and 70% ethyl alcohol to be more effective than plain soap in removing pants included in the studies. Ethics approval 238 Infect Dis Ther (2018) 7:235–247 was not required as EN studies of this type are 7 days prior to testing and during the whole covered by a waiver by the Federal Institute for study. Drugs and Medical Devices (BfArM) in place in Germany. Experimental Design Test Organisms The same study design based on EN1499 was used for both studies, with adaptations for viral Escherichia coli K 12, NCTC 10538, was used as testing. Randomization to treatment sequence the test organism in the bactericidal study. was performed using a Latin Square design. E. coli was chosen, as specified by EN1499, since Each volunteer tested each hand cleansing this is the organism that the EN1499 method is agent and the reference soft soap at 3 and 5 mL validated for in every respect, including volun- volumes, with each volume tested after washing teers’ safety. The contamination fluid used for for 15, 30 and 60 s. Testing was repeated weekly, each test contained between 2.19 9 10 and with volunteers testing all three hand cleansing 5.10 9 10 colony-forming units (cfu)/mL agents at one volume and application time on E. coli. each of six test days. At the end of the whole MNV Berlin, strain S99 was used as the test series, every volunteer had tested each product, organism in the virucidal study. MNV was volume and application time once. chosen as the test virus due to the absence of pathogenicity for humans. The host cells used Bactericidal Test Method for virus cultivation were RAW 264.7 (murine macrophage cell line). The titers of MNV pre- The bactericidal test method is summarized in sent in the test suspensions ranged from 7.67 to Fig. 1. Testing was performed at 20 ± 1 C. 8.83 log tissue culture infectious dose 50% Hands were prepared by washing for 60 s with 5 (TCID )/mL. mL of soft soap to remove transient bacteria and dried with paper towels. Hands were immersed Hand-Cleansing Agents in the contamination fluid for 5 s and then allowed to dry in the air for 3 min, avoiding the The test hand cleansing agents in both studies formation of droplets. Pre-washing samples were PVP-I 7.5% w/v scalp and skin cleanser were obtained by rubbing the fingertips and (Mundipharma Pharmaceuticals, Nicosia, thumb tips on the base of a Petri dish contain- Cyprus) and chlorhexidine gluconate 4% w/v ing 10 mL tryptic soy broth (TSB, without neu- hand cleanser (Mo ¨ lnlycke Health Care US, tralizer) for 60 s. A separate dish was used for -3 -4 Norcross, GA, USA). The reference hand wash each hand. Dilutions of 10 and 10 were procedure used plain soft soap (Sapo kalinus, prepared in TSB and 0.1 mL of each dilution was Pharm. Eur.) 20% w/v. Hand cleansing agents plated on the surface of a tryptic soy agar (TSA) were tested undiluted. plate and incubated at 36 C ± 1 C for 24 h. Volunteers then washed their wetted hands with test product or reference soft soap follow- Healthy Volunteers ing the standard hand wash procedure specified in EN1499. According to EN1499, the standard A total of 15 healthy adult volunteers were hand wash procedure was completed by a 10-s screened and enrolled in each study. All vol- rinse of the hands under cold running tap unteers were in general good health with nor- water. Volunteers held their hands with finger- mal skin (free of dermatoses, cuts, lesions, tips pointing upwards until post-washing sam- hangnails and other skin disorders), short fin- ples were taken by rubbing the fingertips and gernails (\ 2 mm in length), no history of skin thumb tips on the base of a Petri dish contain- disease and had not used oral or topical antibi- ing 10 mL of TSB (with neutralizer) for 60 s. A otics within the previous 2 weeks. All volunteers separate dish was used for each hand. Amounts abstained from using antimicrobial products for Infect Dis Ther (2018) 7:235–247 239 Fig. 1 Bactericidal hygienic hand wash test method and requirements (based on EN1499) of 1.0 and 0.1 mL of the undiluted sampling and incubated at 36 ± 1 C for 24 h. Neutralizer -1 fluid and 0.1 mL of a 10 dilution were pre- XXXII (4% Tween80 ? 3% saponin ? 2% his- pared in TSB, plated on the surface of TSA plates tidine ? 0.4% lecithin ? 0.5% SDS) was used 240 Infect Dis Ther (2018) 7:235–247 for CHG and Neutralizer XLIV ? 2% sodium virus titers were expressed TCID /mL. The thiosulfate (6% Tween80 ? 0.2% his- virucidal activity was determined by the differ- tidine ? 0.6% lecithin ?2% sodium-thiosul- ence of the logarithmic titer of the pre-washing fate ? 0.2% peptone ? 1.7% sodium sample minus the logarithmic titre of the post- chloride ? 1.8% di-sodium hydrogen phos- washing sample (Dlog TCID /mL). This dif- 10 50 phate ? 0.3% potassium dihydrogen phos- ference was given as a reduction factor. phate) was used for PVP-I. The number of test organisms released from Statistical Analysis the fingertips into sampling fluids was assessed before and after the hygienic hand wash and The primary endpoint for each study was the the number of viable bacteria were expressed as mean log reduction factor of the test organism cfu per mL. The bactericidal activity was deter- after the hand wash with each product at each mined by the difference of the mean log pre- pre-defined application time (15, 30 or 60 s) and washing value minus the mean log post-wash- product volume (3 or 5 mL). For testing the data ing value. This difference was given as a reduc- obtained in a Latin-square design experiment tion factor. (where the results of more than one test treat- ment are compared), the statistical analysis methods proposed in EN1499 [comparison of Virucidal Test Method test treatments with a reference treatment (5 mL soft soap for 60 s) in a pairwise manner The virucidal test method is summarized in using a Wilcoxon–Wilcox multiple comparisons Fig. 2. Testing was performed at 20.0 ± 0.5 C. test] were applied, although used in an To remove transient viruses, 5 mL of diluted soft exploratory manner for the virucidal study. The pass criterion for EN1499 was a significance of soap was applied to the cupped hands of vol- unteers and was rubbed in for 60 s before rins- the observed differences between the test and ing with tap water and drying with paper towels reference of p B 0.01. The two test treatments for 30 s. Hands were inoculated with a low were also compared using the same method in concentration suspension of MNV. Volunteers an exploratory manner. The sample size were asked to cup their hands carefully, 250 lL (n = 15) was based on the recommendation of inoculate was slowly pipetted into the palms, given in EN1499. then volunteers gently rubbed their hands together until all surfaces of the hands were RESULTS coated with the inoculate. Finally, volunteers rotated their hands in the air for 60 s to allow Bactericidal Testing the inoculate to dry. Pre-washing samples were obtained by rubbing the fingertips of the inoc- ulated hands on the base of a Petri dish con- The reference hand wash procedure with 5 mL soft soap for 60 s resulted in a mean log taining 5 mL Dulbeccos Minimum Essential Medium for 60 s, followed by drying hands in reduction of E. coli of 2.91. The mean log reductions achieved with the test products PVP- the air for 60 s. Volunteers then washed their hands with the test product or reference soft I and CHG were statistically significantly greater (p B 0.01) than the reduction achieved with the soap following the standard hand wash proce- dure specified in EN1499 before post-washing reference soft soap for all product volumes and contact times (Table 1). There was no statisti- samples were obtained using the same methods cally significant difference in mean log used pre-washing. The Petri dishes were incu- bated at 37 ± 1 C under 5% CO . reduction factors between the different hand washing times using soft soap at either volume The number of test organisms released from the fingertips into sampling fluids was assessed (3 or 5 mL). A small trend towards a greater bacterial reduction with longer washing times before and after the hygienic hand wash, and Infect Dis Ther (2018) 7:235–247 241 Fig. 2 Virucidal hygienic hand wash test method and requirements (as adapted From EN1499) was observed for both test products (except no statistically significant difference between 3 mL CHG; Table 1). An exploratory compar- the two test products for all volumes and time ison of PVP-I and CHG showed that there was points (p [ 0.01) (Table 1). 242 Infect Dis Ther (2018) 7:235–247 Table 1 Results of practical hand wash test with Escherichia coli according to EN1499 Test parameter Reference soft soap PVP-I CHG Product Contact Pre- Post- Log Pre- Post Log Pre- Post- Log 10 10 10 a a a volume time (s) value value reduction value value reduction value value reduction b b 3 mL 15 6.05 3.24 2.80 5.99 1.90 4.09 6.03 0.81 5.22 b b 30 5.93 3.10 2.83 5.94 1.45 4.48 5.95 0.86 5.09 b b 60 5.82 2.96 2.86 5.91 1.12 4.97 6.00 0.86 5.14 b b 5 mL 15 6.02 2.90 3.11 5.93 1.71 4.22 6.00 1.87 4.12 b b 30 5.89 3.14 2.75 5.81 1.49 4.32 5.97 1.64 4.33 b b 60 5.96 3.05 2.91 5.94 0.67 5.27 5.86 1.23 4.63 Each value represents the mean from 30 samples (left and right hands of 15 volunteers) CHG chlorhexidine gluconate 4% hand cleanser, PVP-I povidone-iodine 7.5% hand cleanser Mean log reduction of post-washing versus pre-washing samples Demonstrated a significant difference versus the reference soft soap at the p = 0.01 level (one-sided), denoting that the test product passes the EN1499 test. The reference procedure with soft soap always used 5 mL soap for 60 s contact time per EN1499 standard Virucidal Testing The reference hand wash procedure with 5 mL soft soap for 60 s resulted in a mean log reduction factor of 1.44. The mean log The experimental design using EN1499 is reduction factor achieved with PVP-I was sig- applicable to viral testing with MNV as dis- nificantly greater than the reduction achieved criminatory and reproducible results were gen- with the reference soft soap (p B 0.01) across all erated (Table 2). tests, except for the application of 3 mL for 15 s, for which the reduction achieved was Table 2 Results of practical hand wash test with murine norovirus according to EN1499 Test parameter Reference soft soap PVP-I CHG Product Contact Pre- Post- Log Pre- Post Log Pre- Post- Log 10 10 10 a a a volume time (s) value value reduction value value reduction value value reduction 3 mL 15 5.46 4.22 1.24 5.66 4.09 1.57 5.52 4.62 0.90 30 6.33 4.71 1.62 6.41 4.28 2.13 6.04 4.87 1.18 60 6.37 4.92 1.45 6.19 3.62 2.57 6.27 4.92 1.34 5 mL 15 6.28 4.88 1.41 6.61 4.62 1.99 6.58 5.30 1.28 30 5.99 4.64 1.35 6.05 4.28 1.78 5.90 4.82 1.08 60 6.27 4.83 1.44 6.00 3.81 2.19 6.12 4.83 1.28 Each value represents the mean from 30 samples (left and right hands of 15 volunteers) CHG chlorhexidine gluconate 4% hand cleanser, PVP-I povidone-iodine 7.5% hand cleanser Mean log reduction of post-washing versus pre-washing samples Demonstrated a significant difference versus the reference soft soap at the p = 0.01 level (one-sided) in the modified EN1499 test Infect Dis Ther (2018) 7:235–247 243 numerically but not statistically superior for calcivirus (a surrogate for norovirus) by Lages PVP-I compared with the reference soft soap et al. also showed a significantly greater log (Table 2). In contrast, the mean log reduction reduction factor for 10% PVP-I antiseptic com- factor with CHG was lower than for the refer- pared with two triclosan-containing soaps and ence soft soap across all tests, and a significant five hand sanitizers after 30 s contact time [38]. reduction for CHG compared with the reference A recent review of in vitro and in vivo virucidal soft soap was not achieved (Table 2). A small activity of ethanol concluded that 80% ethanol trend towards a greater viral reduction with is highly effective against enveloped viruses, longer washing times was observed for both test MNV and adenovirus type 5 are usually inacti- products (except 5 mL CHG) and soft soap vated by 70–90% ethanol, while 95% ethanol is (Table 2). An exploratory comparison of PVP-I required to inactivate most other non-en- and CHG showed that the mean log reduction veloped viruses [39]. factor achieved with PVP-I was significantly Based on the results of our studies and the greater than that achieved with CHG (p B 0.01) in vivo studies discussed above, the antimicro- at both 3 and 5 mL and across all application bial efficacy of PVP-I appears consistently better times. than plain soap, whereas CHG performs worse than plain soap against viruses. The soap com- ponent of the CHG-containing cleanser should Safety and Validation have mechanically reduced the viral count down to a similar level as the reference product, No adverse events were observed or reported which may go beyond efficacy gaps. The bica- during or after either study. Controls and vali- tionic character of the CHG molecule, that is dations in both studies conformed to EN1499 responsible for adhesion to surfaces and reten- requirements. tion of other components as observed in oral use [40, 41], may retain viruses too. This reten- tion phenomenon is not only seen with viruses DISCUSSION [42] but also with some Gram-negative bacteria such as A. baumannii [31]. It should be noted The results of our bactericidal study confirm the that, due to the established cumulative effect of positive results with respect to efficacy of PVP-I CHG [2, 3], repeated use may result in greater and CHG versus plain soap against E. coli shown activity than shown in single-use studies, in previous fingertip contamination studies although repeated use of CHG may also increase [32, 36]. However, PVP-I (7.5 or 10%) has been its known risk of development of resistance shown to achieve significantly higher removal [2, 29, 43]. In contrast, PVP-I demonstrates less rates than CHG 4% and plain soap against other persistent activity [2, 3], but is not associated bacteria, including Staphylococcus aureus, MRSA with development of resistance [44]. and A. baumannii [30, 31, 33, 36]. It should be The method set-up of the EN1499 standard noted that alcohol rubs have outperformed can be considered more robust than the ASTM E PVP-I and CHG in removing E. coli in some method, since it employs an internal standard studies [32, 36, 37]. (i.e., plain soft soap, 5 mL applied for 60 s) with The findings of our virucidal study are in defined success criteria, instead of measuring agreement with the results of a previous study log reductions alone for pass criteria. Log by Steinmann et al. [35] that used MNV as a test reductions alone are subject to inherent vari- organism in a modified finger pad test based on ability in such complex biological systems as the ASTM E 1838 [34], with modifications concentration of infected cells in the inoculate derived from the EN1500 [11]. Steinmann et al. and the details of the individual hand wash found that a PVP-I formulation with procedure. The use of an internal standard also 0.75–0.81% available iodine performed better allows assessment of the biocidal power of the than three alcohol-based hand rubs, while 4% test product versus the purely mechanical/de- CHG and 1% triclosan hand washes were inef- tergent-based removal of test organisms by non- fective [35]. An earlier fingertip test using feline 244 Infect Dis Ther (2018) 7:235–247 medicated soft soap. Additionally, the EN1499 be taken into consideration given the limita- method has more power to demonstrate efficacy tions of in vivo studies. of the products tested due to the larger sample Another limitation of current in vivo stan- size (15 volunteers) compared with the ASTM E dard test methods, including EN1499, is that method and several previous fingertip bacteri- they do not account for extrinsic factors such as cidal studies [30, 31, 36], which include 5 vol- protein load on soiled hands, residual activity (a unteers. The hand wash procedure was notable characteristic of CHG [2]) and how the standardized as much as possible in our study product is used/applied by individuals [3]. It by following the CEN procedure (in the mean- should also be emphasized that the degree of while adapted by WHO) closely, and shorter reduction in microbial counts required to pro- contact times of 15 and 30 s were included to duce a meaningful drop in the hand-borne more closely reflect real-life use [45–47]. The use spread of nosocomial pathogens has yet to be of various volumes and exposure times also quantified, and thus the clinical relevance of provided further validation of the internal ref- such in vivo test results remains unclear erence used. We have shown for the first time in [23, 24]. Controlled clinical, observational and this study that the bacterial testing from epidemiological studies are thus needed for EN1499 can be adapted to MNV-based viral more direct proof of clinical effectiveness, but testing and may be a good basis for further are generally lacking, although PVP-I hand virucidal CEN standards, pending further vali- scrub has previously been shown to have supe- dation as interlaboratory tests. It may be rior efficacy to soap and water in a neonatal hypothesized that antiseptic products which are intensive care unit [50]. effective against non-enveloped viruses such as Another important consideration in selec- MNV will be effective against enveloped viruses tion of hand hygiene agents, particularly in such as Ebola (EBOV), severe acute respiratory resource-poor countries, is cost and availability. syndrome and Middle East respiratory syn- Alcohol-based hand rub formulations proposed drome coronaviruses (SARS- and MERS-CoV), by the WHO have recently been shown to be influenza and other emerging viruses; this is effective against enveloped viruses including supported by the results of in vitro suspension emerging Zika, EBOV, SARS-CoV and MERS- tests with the modified vaccinia virus Ankara CoV in vitro [51], and may be a more freely [12, 13]. available and cost-effective alternative to med- An obvious limitation of our studies is the icated cleansers/antiseptics. Since hand rubs use of only a single test pathogen (E. coli and cannot properly cleanse soiled hands [2], a MNV). In vivo testing is necessarily restricted to phased approach as recommended by the medically safe organisms to minimize the risk to National Institute for Health and Care Excel- healthy human volunteers, and therefore E. coli lence [52] may be an optimized approach to (strain NCTC 10538) was chosen as the model handle situations where increased hand pathogen for EN1499. ASTM E1174 [48], the hygiene is warranted. only other standard in vivo method employing the whole hands of human subjects, similarly CONCLUSION suggests a single test organism (primarily, Ser- ratia marcescens, or alternatively, E. coli strain In conclusion, in these simulated hand wash ATCC 11229). ASTM E2276 [49] allows for using studies, PVP-I 7.5% scalp and skin cleanser a variety of medically relevant test species (S. passed the requirements of EN1499 (adapted for marcescens , E. coli, A. baumannii, S. aureus, Sta- viruses) and showed significantly better efficacy phylococcus epidermidis, Candida albicans, Asper- against E. coli and MNV than the reference soft gillus niger), but confines testing to the finger soap within 15 s using a 3-mL application pads rather than the whole hands and only (E. coli) or a 5-mL application (MNV). CHG 4% assesses the simple removal of test organisms hand cleanser also passed the requirements of from the hands with no internal reference. EN1499 against E. coli within 15 s using a 3-mL In vitro data on potential efficacy gaps [2] can Infect Dis Ther (2018) 7:235–247 245 application, but was ineffective against MNV an employee of Mundipharma Research Lim- compared to hand washing with soft soap. ited. Craig Newby is an employee of Mundi- Although the results have yet to be confirmed pharma Manufacturing Pte Ltd. Stefan Mu ¨ ller is in clinical and epidemiological studies, these an employee of Mundipharma Research GmbH studies provide important public health infor- & Co.KG. mation for the appropriate use of hand hygiene Compliance with Ethics Guidelines. All products. procedures performed in studies involving human participants were in accordance with the 1964 Helsinki declaration and its later ACKNOWLEDGEMENTS amendments or comparable ethical standards. Informed consent was obtained from all indi- We thank the participants of the studies. Special vidual participants included in the studies. thanks are given to Carolin Benzinger, Sarah Ethics approval was not required as EN studies Bachmeier and Birgit Ahrens who helped con- of this type are covered by a waiver by the duct the study at the Laboratory Enders, for Federal Institute for Drugs and Medical Devices their excellent technical assistance. (BfArM) in place in Germany. Funding. Sponsorship for both studies and Open Access. This article is distributed article processing charges was funded by Mun- under the terms of the Creative Commons dipharma Manufacturing Pte Ltd. All authors Attribution-NonCommercial 4.0 International had full access to all of the data in this study License (http://creativecommons.org/licenses/ and take complete responsibility for the integ- by-nc/4.0/), which permits any noncommercial rity of the data and accuracy of the data use, distribution, and reproduction in any analysis. medium, provided you give appropriate credit to the original author(s) and the source, provide Editorial Assistance. Editorial assistance in a link to the Creative Commons license, and the preparation of this article was provided by indicate if changes were made. Karen Mower of Scientific Editorial. Support for this assistance was funded by Mundipharma Research GmbH & Co.KG. REFERENCES Authorship. All named authors meet the International Committee of Medical Journal 1. Kampf G, Kramer A. 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Bactericidal and Virucidal Activity of Povidone-Iodine and Chlorhexidine Gluconate Cleansers in an In Vivo Hand Hygiene Clinical Simulation Study

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Medicine & Public Health; Internal Medicine; Infectious Diseases
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Abstract

Infect Dis Ther (2018) 7:235–247 https://doi.org/10.1007/s40121-018-0202-5 ORIGINAL RESEARCH Bactericidal and Virucidal Activity of Povidone-Iodine and Chlorhexidine Gluconate Cleansers in an In Vivo Hand Hygiene Clinical Simulation Study . . . Maren Eggers Torsten Koburger-Janssen Lois S. Ward Craig Newby Stefan Mu ¨ ller Received: February 6, 2018 / Published online: May 14, 2018 The Author(s) 2018 method for bacteria), which was adapted for ABSTRACT virucidal testing. Methods: Separate test series were performed Introduction: Standard in vitro and in vivo for bactericidal (Escherichia coli) and virucidal tests help demonstrate efficacy of hand hygiene [murine norovirus (MNV)] testing. After pre- products; however, there is no standard in vivo washing and artificial contamination of hands test method for viruses. We investigated the with test organisms, volunteers underwent bactericidal and virucidal efficacy of povidone- testing with 3 and 5 mL of each product for iodine (PVP-I) 7.5% scalp and skin cleanser, contact times of 15, 30 and 60 s according to a chlorhexidine gluconate (CHG) 4% hand Latin-square randomization. The number of test cleanser and the reference hand wash (soft soap) organisms released from fingertips into sam- in 15 healthy volunteers following European pling fluids was assessed before and after hand Standard EN1499 (hygienic hand wash test washing and mean log reduction factor (RF) was calculated. RFs (test-reference) were com- Enhanced Digital Features To view enhanced digital pared using a Wilcoxon–Wilcox multiple com- features for this article go to https://doi.org/10.6084/ parisons test per EN1499; efficacy was m9.figshare.6200894. concluded if p B 0.01. Results: PVP-I 7.5% and CHG 4% cleansers M. Eggers both passed EN1499 requirements against Labor Prof Gisela Enders MVZ GbR, Stuttgart, E. coli, with statistically significantly greater (p Germany B 0.01) mean log RFs compared with refer- T. Koburger-Janssen ence soft soap across all tests (PVP-I: 4.09–5.27; Hygiene Nord GmbH, Greifswald, Germany CHG: 4.12–5.22; soap: 2.75–3.11). The experi- L. S. Ward mental design using EN1499 was applicable to Clinical Operations, Mundipharma Research testing with MNV as discriminatory and repro- Limited, Cambridge, UK ducible results were generated. Mean log RFs C. Newby of MNV were statistically significantly greater R&D, Mundipharma Manufacturing Pte Ltd, for PVP-I (1.57–2.57) compared with soft soap Singapore, Singapore (1.24–1.62), while mean log RFs with CHG S. Mu ¨ ller (&) (0.90–1.34) were lower than for soft soap across Pharmacologicial and Translational Science, all tests. Mundipharma Research GmbH & Co KG, Limburg, Conclusion: PVP-I 7.5% cleanser showed supe- Germany rior efficacy against MNV compared to soft soap e-mail: stefan.mueller@mundipharma-rd.eu 236 Infect Dis Ther (2018) 7:235–247 and CHG 4% cleanser, while both PVP-I and effective than plain soap at removing viruses in CHG were superior to soft soap against E. coli. all tests. The adapted test method using murine The experimental set-up may be applicable to norovirus worked well, giving consistent results future testing for antiviral hand washes. for each product, and may be suitable for future Funding: Mundipharma Manufacturing Pte testing of antiseptic products against viruses. Ltd. Plain Language Summary: Plain language INTRODUCTION summary available for this article. The importance of hand hygiene in health care Keywords: Antimicrobial wash; Bactericidal and community settings is undoubted, with activity; Chlorhexidine gluconate; Escherichia numerous studies demonstrating the associa- coli; Hand-cleansing agents; Hand hygiene; tion between proper hand hygiene and reduc- Infection control; Non-enveloped virus; tions in both rates of nosocomial infections Povidone-iodine; Virucidal activity [1–3] and rates of infectious illnesses in com- munities [4]. Hand hygiene is also an infection control procedure that can be rapidly adopted PLAIN LANGUAGE SUMMARY by the public and healthcare workers in the case of emerging and re-emerging infectious diseases Serious infectious disease outbreaks and hospi- to limit the spread of viruses by breaking the tal-acquired infections are threats to human transmission [5, 6]. health. Effective vaccines and drug products are Multiple hand hygiene agents are currently often not available, but antiseptic products for available including plain non-medicated soap, hand washing can help to control the spread of medicated hand washes with various active disease. Standard methods are used to test the ingredients, and waterless or alcohol-based effectiveness of hand hygiene products in the hand rubs. Although the use of alcohol-based laboratory and on the hands of human hand rubs is convenient, the World Health volunteers. Organization (WHO) recommends washing We used a standard hand washing test hands with soap and water when visibly dirty or method to find out whether two antiseptic visibly soiled with blood or other body fluids products work against bacteria (Escherichia coli). [2]. It is recognized that some antiseptics and The method was adapted to also test effective- alcohol-based hand rubs are characterized by ness against viruses (murine norovirus). The resistant bacteria and efficacy gaps [1–3]. products tested were povidone-iodine scalp and In vitro suspension tests and human challenge skin cleanser, and chlorhexidine gluconate trials help to demonstrate which hand hygiene hand cleanser, which were compared to plain agents are fit for purpose, but variations in soft soap. After contamination of hands with methodology affect the measurement of effi- the bacteria/virus, volunteers washed their cacy [7]. Ensuring that hand rub and hand wash hands for 15, 30 or 60 s, using 3 or 5 mL of products pass standardized antimicrobial activ- product, in separate tests. Bacteria/viruses pre- ity tests helps to better understand and interpret sent on volunteers’ hands were measured before efficacy results [7]. and after hand washing. The antiseptic product In Europe, the most commonly used meth- had to remove significantly more bacteria/ ods to test hand antiseptics are those of the viruses than plain soap to be rated as effective. European Committee for Standardization Povidone-iodine cleanser and chlorhexidine (CEN), while in the USA and Canada, the stan- gluconate cleanser were both more effective dards of ASTM International (formerly, the than plain soap at removing bacteria from vol- American Society for Testing and Materials) are unteers’ hands. Povidone-iodine cleanser was used. The CEN has adopted a hierarchical, sys- more effective than plain soap at removing tematic approach of product testing. In this viruses, except when using 3 mL product for concept, European Standards EN1276 and 15 s. Chlorhexidine gluconate cleanser was less Infect Dis Ther (2018) 7:235–247 237 EN14476 are established initial in vitro methods methicillin-resistant Staphylococcus aureus to determine the bactericidal and virucidal (MRSA), Acinetobacter baumannii and E. coli from efficacy of antiseptics and disinfectants [8, 9]. volunteers’ hands, while CHG was only signifi- The next step in the CEN testing scheme is cantly more effective than plain soap at European standard EN1499 (for hand washes) removing E. coli [30–33]. In vivo virucidal and EN1500 (for hand rubs), each Phase 2/Step studies are generally lacking; however, in a 2 hand simulation studies where the hands of modified finger pad test based on the ASTM E volunteers are artificially contaminated with 1838 [34] using MNV, PVP-I was shown to have E. coli and the test product is compared against a superior efficacy to three alcohol-based hand reference procedure in a crossover design rubs while 4% CHG and 1% triclosan showed [10, 11]. To fill in gaps within the in vitro CEN no activity [35]. testing, modified vaccinia virus Ankara (MVA) We undertook two hand hygiene in vivo was introduced in 2015 as a reference virus for clinical simulation studies to investigate the the claim of virucidal activity against enveloped bactericidal and virucidal efficacy of PVP-I 7.5% viruses for hygienic hand rub and hand wash scalp and skin cleanser and CHG 4% hand products [9, 12, 13]. However, it has not been cleanser versus the reference hand wash (soft possible to develop a Phase 2/Step 2 test for soap) on healthy volunteers’ hands. Both stud- enveloped viruses due to the lack of availability ies followed European standard EN1499 as a of a safe enveloped virus that can be ethically function of employed soap volume and hand used in testing on volunteers’ hands, and there washing time. The virucidal study was per- is currently no standard in vivo test method for formed in an exploratory manner using murine viruses. To fill this gap, a hand simulation test norovirus (MNV) as model test virus. has recently been considered that uses murine norovirus (MNV), a non-pathogenic (biosafety METHODS level 1) non-enveloped virus, which is more resilient to antiseptics than enveloped viruses, Study Design as a model test organism. Povidone-iodine (PVP-I) and chlorhexidine Each study was randomized, controlled, open gluconate (CHG) are broad-spectrum antimi- crobials that have been used in infection con- label, crossover and exploratory in design. The bactericidal study was conducted at the labora- trol and prevention for more than 60 years. PVP-I has well-established general antimicrobial tories of Hygiene Nord, Greifswald, Germany, from 1 September 2016 to 19 January 2017. The activity, particularly in relation to resistant organisms [2], demonstrating in vitro activity virucidal study was conducted at the laborato- ries of Labor Prof. Gisela Enders MVZ GbR, against Gram-positive, Gram-negative and some spore-forming bacteria (Clostridia, Bacillus Stuttgart, Germany, from 14 September 2016 to spp.) and mycobacteria [2, 14–18] and a wide 12 December 2016. Hand hygiene tests were performed on the hands of healthy volunteers range of enveloped and non-enveloped viruses [12, 13, 19–22]. In contrast, CHG has good simulating practical use according to the methods described in European Standard in vitro activity against Gram-positive bacteria and enveloped viruses, less activity against EN1499:2013 (hygienic hand wash test method for bacteria), which were adapted and used in Gram-negative bacteria and non-enveloped viruses, and minimal activity against mycobac- an exploratory manner to assess the virucidal activity of the hand cleansers. All procedures teria [22–28]. CHG resistance is a concern and has been detected in isolates of Enterobacter spp., performed in studies involving human partici- pants were in accordance with the 1964 Hel- Pseudomonas spp., Proteus spp., Providencia spp. sinki declaration and its later amendments or and Enterococcus spp [29]. In vivo studies using artificial fingertip contamination have previ- comparable ethical standards. Informed con- sent was obtained from all individual partici- ously shown PVP-I and 70% ethyl alcohol to be more effective than plain soap in removing pants included in the studies. Ethics approval 238 Infect Dis Ther (2018) 7:235–247 was not required as EN studies of this type are 7 days prior to testing and during the whole covered by a waiver by the Federal Institute for study. Drugs and Medical Devices (BfArM) in place in Germany. Experimental Design Test Organisms The same study design based on EN1499 was used for both studies, with adaptations for viral Escherichia coli K 12, NCTC 10538, was used as testing. Randomization to treatment sequence the test organism in the bactericidal study. was performed using a Latin Square design. E. coli was chosen, as specified by EN1499, since Each volunteer tested each hand cleansing this is the organism that the EN1499 method is agent and the reference soft soap at 3 and 5 mL validated for in every respect, including volun- volumes, with each volume tested after washing teers’ safety. The contamination fluid used for for 15, 30 and 60 s. Testing was repeated weekly, each test contained between 2.19 9 10 and with volunteers testing all three hand cleansing 5.10 9 10 colony-forming units (cfu)/mL agents at one volume and application time on E. coli. each of six test days. At the end of the whole MNV Berlin, strain S99 was used as the test series, every volunteer had tested each product, organism in the virucidal study. MNV was volume and application time once. chosen as the test virus due to the absence of pathogenicity for humans. The host cells used Bactericidal Test Method for virus cultivation were RAW 264.7 (murine macrophage cell line). The titers of MNV pre- The bactericidal test method is summarized in sent in the test suspensions ranged from 7.67 to Fig. 1. Testing was performed at 20 ± 1 C. 8.83 log tissue culture infectious dose 50% Hands were prepared by washing for 60 s with 5 (TCID )/mL. mL of soft soap to remove transient bacteria and dried with paper towels. Hands were immersed Hand-Cleansing Agents in the contamination fluid for 5 s and then allowed to dry in the air for 3 min, avoiding the The test hand cleansing agents in both studies formation of droplets. Pre-washing samples were PVP-I 7.5% w/v scalp and skin cleanser were obtained by rubbing the fingertips and (Mundipharma Pharmaceuticals, Nicosia, thumb tips on the base of a Petri dish contain- Cyprus) and chlorhexidine gluconate 4% w/v ing 10 mL tryptic soy broth (TSB, without neu- hand cleanser (Mo ¨ lnlycke Health Care US, tralizer) for 60 s. A separate dish was used for -3 -4 Norcross, GA, USA). The reference hand wash each hand. Dilutions of 10 and 10 were procedure used plain soft soap (Sapo kalinus, prepared in TSB and 0.1 mL of each dilution was Pharm. Eur.) 20% w/v. Hand cleansing agents plated on the surface of a tryptic soy agar (TSA) were tested undiluted. plate and incubated at 36 C ± 1 C for 24 h. Volunteers then washed their wetted hands with test product or reference soft soap follow- Healthy Volunteers ing the standard hand wash procedure specified in EN1499. According to EN1499, the standard A total of 15 healthy adult volunteers were hand wash procedure was completed by a 10-s screened and enrolled in each study. All vol- rinse of the hands under cold running tap unteers were in general good health with nor- water. Volunteers held their hands with finger- mal skin (free of dermatoses, cuts, lesions, tips pointing upwards until post-washing sam- hangnails and other skin disorders), short fin- ples were taken by rubbing the fingertips and gernails (\ 2 mm in length), no history of skin thumb tips on the base of a Petri dish contain- disease and had not used oral or topical antibi- ing 10 mL of TSB (with neutralizer) for 60 s. A otics within the previous 2 weeks. All volunteers separate dish was used for each hand. Amounts abstained from using antimicrobial products for Infect Dis Ther (2018) 7:235–247 239 Fig. 1 Bactericidal hygienic hand wash test method and requirements (based on EN1499) of 1.0 and 0.1 mL of the undiluted sampling and incubated at 36 ± 1 C for 24 h. Neutralizer -1 fluid and 0.1 mL of a 10 dilution were pre- XXXII (4% Tween80 ? 3% saponin ? 2% his- pared in TSB, plated on the surface of TSA plates tidine ? 0.4% lecithin ? 0.5% SDS) was used 240 Infect Dis Ther (2018) 7:235–247 for CHG and Neutralizer XLIV ? 2% sodium virus titers were expressed TCID /mL. The thiosulfate (6% Tween80 ? 0.2% his- virucidal activity was determined by the differ- tidine ? 0.6% lecithin ?2% sodium-thiosul- ence of the logarithmic titer of the pre-washing fate ? 0.2% peptone ? 1.7% sodium sample minus the logarithmic titre of the post- chloride ? 1.8% di-sodium hydrogen phos- washing sample (Dlog TCID /mL). This dif- 10 50 phate ? 0.3% potassium dihydrogen phos- ference was given as a reduction factor. phate) was used for PVP-I. The number of test organisms released from Statistical Analysis the fingertips into sampling fluids was assessed before and after the hygienic hand wash and The primary endpoint for each study was the the number of viable bacteria were expressed as mean log reduction factor of the test organism cfu per mL. The bactericidal activity was deter- after the hand wash with each product at each mined by the difference of the mean log pre- pre-defined application time (15, 30 or 60 s) and washing value minus the mean log post-wash- product volume (3 or 5 mL). For testing the data ing value. This difference was given as a reduc- obtained in a Latin-square design experiment tion factor. (where the results of more than one test treat- ment are compared), the statistical analysis methods proposed in EN1499 [comparison of Virucidal Test Method test treatments with a reference treatment (5 mL soft soap for 60 s) in a pairwise manner The virucidal test method is summarized in using a Wilcoxon–Wilcox multiple comparisons Fig. 2. Testing was performed at 20.0 ± 0.5 C. test] were applied, although used in an To remove transient viruses, 5 mL of diluted soft exploratory manner for the virucidal study. The pass criterion for EN1499 was a significance of soap was applied to the cupped hands of vol- unteers and was rubbed in for 60 s before rins- the observed differences between the test and ing with tap water and drying with paper towels reference of p B 0.01. The two test treatments for 30 s. Hands were inoculated with a low were also compared using the same method in concentration suspension of MNV. Volunteers an exploratory manner. The sample size were asked to cup their hands carefully, 250 lL (n = 15) was based on the recommendation of inoculate was slowly pipetted into the palms, given in EN1499. then volunteers gently rubbed their hands together until all surfaces of the hands were RESULTS coated with the inoculate. Finally, volunteers rotated their hands in the air for 60 s to allow Bactericidal Testing the inoculate to dry. Pre-washing samples were obtained by rubbing the fingertips of the inoc- ulated hands on the base of a Petri dish con- The reference hand wash procedure with 5 mL soft soap for 60 s resulted in a mean log taining 5 mL Dulbeccos Minimum Essential Medium for 60 s, followed by drying hands in reduction of E. coli of 2.91. The mean log reductions achieved with the test products PVP- the air for 60 s. Volunteers then washed their hands with the test product or reference soft I and CHG were statistically significantly greater (p B 0.01) than the reduction achieved with the soap following the standard hand wash proce- dure specified in EN1499 before post-washing reference soft soap for all product volumes and contact times (Table 1). There was no statisti- samples were obtained using the same methods cally significant difference in mean log used pre-washing. The Petri dishes were incu- bated at 37 ± 1 C under 5% CO . reduction factors between the different hand washing times using soft soap at either volume The number of test organisms released from the fingertips into sampling fluids was assessed (3 or 5 mL). A small trend towards a greater bacterial reduction with longer washing times before and after the hygienic hand wash, and Infect Dis Ther (2018) 7:235–247 241 Fig. 2 Virucidal hygienic hand wash test method and requirements (as adapted From EN1499) was observed for both test products (except no statistically significant difference between 3 mL CHG; Table 1). An exploratory compar- the two test products for all volumes and time ison of PVP-I and CHG showed that there was points (p [ 0.01) (Table 1). 242 Infect Dis Ther (2018) 7:235–247 Table 1 Results of practical hand wash test with Escherichia coli according to EN1499 Test parameter Reference soft soap PVP-I CHG Product Contact Pre- Post- Log Pre- Post Log Pre- Post- Log 10 10 10 a a a volume time (s) value value reduction value value reduction value value reduction b b 3 mL 15 6.05 3.24 2.80 5.99 1.90 4.09 6.03 0.81 5.22 b b 30 5.93 3.10 2.83 5.94 1.45 4.48 5.95 0.86 5.09 b b 60 5.82 2.96 2.86 5.91 1.12 4.97 6.00 0.86 5.14 b b 5 mL 15 6.02 2.90 3.11 5.93 1.71 4.22 6.00 1.87 4.12 b b 30 5.89 3.14 2.75 5.81 1.49 4.32 5.97 1.64 4.33 b b 60 5.96 3.05 2.91 5.94 0.67 5.27 5.86 1.23 4.63 Each value represents the mean from 30 samples (left and right hands of 15 volunteers) CHG chlorhexidine gluconate 4% hand cleanser, PVP-I povidone-iodine 7.5% hand cleanser Mean log reduction of post-washing versus pre-washing samples Demonstrated a significant difference versus the reference soft soap at the p = 0.01 level (one-sided), denoting that the test product passes the EN1499 test. The reference procedure with soft soap always used 5 mL soap for 60 s contact time per EN1499 standard Virucidal Testing The reference hand wash procedure with 5 mL soft soap for 60 s resulted in a mean log reduction factor of 1.44. The mean log The experimental design using EN1499 is reduction factor achieved with PVP-I was sig- applicable to viral testing with MNV as dis- nificantly greater than the reduction achieved criminatory and reproducible results were gen- with the reference soft soap (p B 0.01) across all erated (Table 2). tests, except for the application of 3 mL for 15 s, for which the reduction achieved was Table 2 Results of practical hand wash test with murine norovirus according to EN1499 Test parameter Reference soft soap PVP-I CHG Product Contact Pre- Post- Log Pre- Post Log Pre- Post- Log 10 10 10 a a a volume time (s) value value reduction value value reduction value value reduction 3 mL 15 5.46 4.22 1.24 5.66 4.09 1.57 5.52 4.62 0.90 30 6.33 4.71 1.62 6.41 4.28 2.13 6.04 4.87 1.18 60 6.37 4.92 1.45 6.19 3.62 2.57 6.27 4.92 1.34 5 mL 15 6.28 4.88 1.41 6.61 4.62 1.99 6.58 5.30 1.28 30 5.99 4.64 1.35 6.05 4.28 1.78 5.90 4.82 1.08 60 6.27 4.83 1.44 6.00 3.81 2.19 6.12 4.83 1.28 Each value represents the mean from 30 samples (left and right hands of 15 volunteers) CHG chlorhexidine gluconate 4% hand cleanser, PVP-I povidone-iodine 7.5% hand cleanser Mean log reduction of post-washing versus pre-washing samples Demonstrated a significant difference versus the reference soft soap at the p = 0.01 level (one-sided) in the modified EN1499 test Infect Dis Ther (2018) 7:235–247 243 numerically but not statistically superior for calcivirus (a surrogate for norovirus) by Lages PVP-I compared with the reference soft soap et al. also showed a significantly greater log (Table 2). In contrast, the mean log reduction reduction factor for 10% PVP-I antiseptic com- factor with CHG was lower than for the refer- pared with two triclosan-containing soaps and ence soft soap across all tests, and a significant five hand sanitizers after 30 s contact time [38]. reduction for CHG compared with the reference A recent review of in vitro and in vivo virucidal soft soap was not achieved (Table 2). A small activity of ethanol concluded that 80% ethanol trend towards a greater viral reduction with is highly effective against enveloped viruses, longer washing times was observed for both test MNV and adenovirus type 5 are usually inacti- products (except 5 mL CHG) and soft soap vated by 70–90% ethanol, while 95% ethanol is (Table 2). An exploratory comparison of PVP-I required to inactivate most other non-en- and CHG showed that the mean log reduction veloped viruses [39]. factor achieved with PVP-I was significantly Based on the results of our studies and the greater than that achieved with CHG (p B 0.01) in vivo studies discussed above, the antimicro- at both 3 and 5 mL and across all application bial efficacy of PVP-I appears consistently better times. than plain soap, whereas CHG performs worse than plain soap against viruses. The soap com- ponent of the CHG-containing cleanser should Safety and Validation have mechanically reduced the viral count down to a similar level as the reference product, No adverse events were observed or reported which may go beyond efficacy gaps. The bica- during or after either study. Controls and vali- tionic character of the CHG molecule, that is dations in both studies conformed to EN1499 responsible for adhesion to surfaces and reten- requirements. tion of other components as observed in oral use [40, 41], may retain viruses too. This reten- tion phenomenon is not only seen with viruses DISCUSSION [42] but also with some Gram-negative bacteria such as A. baumannii [31]. It should be noted The results of our bactericidal study confirm the that, due to the established cumulative effect of positive results with respect to efficacy of PVP-I CHG [2, 3], repeated use may result in greater and CHG versus plain soap against E. coli shown activity than shown in single-use studies, in previous fingertip contamination studies although repeated use of CHG may also increase [32, 36]. However, PVP-I (7.5 or 10%) has been its known risk of development of resistance shown to achieve significantly higher removal [2, 29, 43]. In contrast, PVP-I demonstrates less rates than CHG 4% and plain soap against other persistent activity [2, 3], but is not associated bacteria, including Staphylococcus aureus, MRSA with development of resistance [44]. and A. baumannii [30, 31, 33, 36]. It should be The method set-up of the EN1499 standard noted that alcohol rubs have outperformed can be considered more robust than the ASTM E PVP-I and CHG in removing E. coli in some method, since it employs an internal standard studies [32, 36, 37]. (i.e., plain soft soap, 5 mL applied for 60 s) with The findings of our virucidal study are in defined success criteria, instead of measuring agreement with the results of a previous study log reductions alone for pass criteria. Log by Steinmann et al. [35] that used MNV as a test reductions alone are subject to inherent vari- organism in a modified finger pad test based on ability in such complex biological systems as the ASTM E 1838 [34], with modifications concentration of infected cells in the inoculate derived from the EN1500 [11]. Steinmann et al. and the details of the individual hand wash found that a PVP-I formulation with procedure. The use of an internal standard also 0.75–0.81% available iodine performed better allows assessment of the biocidal power of the than three alcohol-based hand rubs, while 4% test product versus the purely mechanical/de- CHG and 1% triclosan hand washes were inef- tergent-based removal of test organisms by non- fective [35]. An earlier fingertip test using feline 244 Infect Dis Ther (2018) 7:235–247 medicated soft soap. Additionally, the EN1499 be taken into consideration given the limita- method has more power to demonstrate efficacy tions of in vivo studies. of the products tested due to the larger sample Another limitation of current in vivo stan- size (15 volunteers) compared with the ASTM E dard test methods, including EN1499, is that method and several previous fingertip bacteri- they do not account for extrinsic factors such as cidal studies [30, 31, 36], which include 5 vol- protein load on soiled hands, residual activity (a unteers. The hand wash procedure was notable characteristic of CHG [2]) and how the standardized as much as possible in our study product is used/applied by individuals [3]. It by following the CEN procedure (in the mean- should also be emphasized that the degree of while adapted by WHO) closely, and shorter reduction in microbial counts required to pro- contact times of 15 and 30 s were included to duce a meaningful drop in the hand-borne more closely reflect real-life use [45–47]. The use spread of nosocomial pathogens has yet to be of various volumes and exposure times also quantified, and thus the clinical relevance of provided further validation of the internal ref- such in vivo test results remains unclear erence used. We have shown for the first time in [23, 24]. Controlled clinical, observational and this study that the bacterial testing from epidemiological studies are thus needed for EN1499 can be adapted to MNV-based viral more direct proof of clinical effectiveness, but testing and may be a good basis for further are generally lacking, although PVP-I hand virucidal CEN standards, pending further vali- scrub has previously been shown to have supe- dation as interlaboratory tests. It may be rior efficacy to soap and water in a neonatal hypothesized that antiseptic products which are intensive care unit [50]. effective against non-enveloped viruses such as Another important consideration in selec- MNV will be effective against enveloped viruses tion of hand hygiene agents, particularly in such as Ebola (EBOV), severe acute respiratory resource-poor countries, is cost and availability. syndrome and Middle East respiratory syn- Alcohol-based hand rub formulations proposed drome coronaviruses (SARS- and MERS-CoV), by the WHO have recently been shown to be influenza and other emerging viruses; this is effective against enveloped viruses including supported by the results of in vitro suspension emerging Zika, EBOV, SARS-CoV and MERS- tests with the modified vaccinia virus Ankara CoV in vitro [51], and may be a more freely [12, 13]. available and cost-effective alternative to med- An obvious limitation of our studies is the icated cleansers/antiseptics. Since hand rubs use of only a single test pathogen (E. coli and cannot properly cleanse soiled hands [2], a MNV). In vivo testing is necessarily restricted to phased approach as recommended by the medically safe organisms to minimize the risk to National Institute for Health and Care Excel- healthy human volunteers, and therefore E. coli lence [52] may be an optimized approach to (strain NCTC 10538) was chosen as the model handle situations where increased hand pathogen for EN1499. ASTM E1174 [48], the hygiene is warranted. only other standard in vivo method employing the whole hands of human subjects, similarly CONCLUSION suggests a single test organism (primarily, Ser- ratia marcescens, or alternatively, E. coli strain In conclusion, in these simulated hand wash ATCC 11229). ASTM E2276 [49] allows for using studies, PVP-I 7.5% scalp and skin cleanser a variety of medically relevant test species (S. passed the requirements of EN1499 (adapted for marcescens , E. coli, A. baumannii, S. aureus, Sta- viruses) and showed significantly better efficacy phylococcus epidermidis, Candida albicans, Asper- against E. coli and MNV than the reference soft gillus niger), but confines testing to the finger soap within 15 s using a 3-mL application pads rather than the whole hands and only (E. coli) or a 5-mL application (MNV). CHG 4% assesses the simple removal of test organisms hand cleanser also passed the requirements of from the hands with no internal reference. EN1499 against E. coli within 15 s using a 3-mL In vitro data on potential efficacy gaps [2] can Infect Dis Ther (2018) 7:235–247 245 application, but was ineffective against MNV an employee of Mundipharma Research Lim- compared to hand washing with soft soap. ited. Craig Newby is an employee of Mundi- Although the results have yet to be confirmed pharma Manufacturing Pte Ltd. Stefan Mu ¨ ller is in clinical and epidemiological studies, these an employee of Mundipharma Research GmbH studies provide important public health infor- & Co.KG. mation for the appropriate use of hand hygiene Compliance with Ethics Guidelines. All products. procedures performed in studies involving human participants were in accordance with the 1964 Helsinki declaration and its later ACKNOWLEDGEMENTS amendments or comparable ethical standards. Informed consent was obtained from all indi- We thank the participants of the studies. Special vidual participants included in the studies. thanks are given to Carolin Benzinger, Sarah Ethics approval was not required as EN studies Bachmeier and Birgit Ahrens who helped con- of this type are covered by a waiver by the duct the study at the Laboratory Enders, for Federal Institute for Drugs and Medical Devices their excellent technical assistance. (BfArM) in place in Germany. Funding. Sponsorship for both studies and Open Access. This article is distributed article processing charges was funded by Mun- under the terms of the Creative Commons dipharma Manufacturing Pte Ltd. All authors Attribution-NonCommercial 4.0 International had full access to all of the data in this study License (http://creativecommons.org/licenses/ and take complete responsibility for the integ- by-nc/4.0/), which permits any noncommercial rity of the data and accuracy of the data use, distribution, and reproduction in any analysis. medium, provided you give appropriate credit to the original author(s) and the source, provide Editorial Assistance. Editorial assistance in a link to the Creative Commons license, and the preparation of this article was provided by indicate if changes were made. Karen Mower of Scientific Editorial. Support for this assistance was funded by Mundipharma Research GmbH & Co.KG. REFERENCES Authorship. All named authors meet the International Committee of Medical Journal 1. Kampf G, Kramer A. 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Infectious Diseases and TherapySpringer Journals

Published: May 14, 2018

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